1. Field of the Invention
The invention relates to laser-induced breakdown spectroscopy (LIBS), and more particularly to an apparatus suitable for conducting microscale LIBS analyses of biological cells, the cell interiors, and the cell environs.
2. Description of the Prior Art
The analytical technique of laser-induced breakdown spectroscopy (LIBS), also called laser-induced plasma spectroscopy (LIPS) or laser plasma spectroscopy (LPS), focuses a high-power laser beam onto a sample surface, with each pulse sparking on and vaporizing a small mass (sub-microgram) of sample and creating a microplasma. As the constituents of the plasma plume de-excite during cooling, they give off light with wavelengths characteristic of the elemental constituents of the plasma. By analyzing this light, the sample contents can be determined and often quantified, without the need for sample preparation or long analytical lead times.
Detection of most of the elements of the periodic table using LIBS has been demonstrated for a variety of samples and sample matrices (solid, liquid, aerosol, and gas phase). For example, LIBS was established at ORNL by the co-inventors and others during the development of in-situ, real-time air monitors for hazardous metals in atmospheric emissions.
Current analyses of pharmaceutical distributions within cells are very tedious. Conventional analyses of average elemental distributions within cells, such as boron distributions between cytoplasm and nucleus, typically involve digestion of the cells, followed by fractionation and separate analyses of nuclear and cytoplasmic components. More expensive analytical techniques such as inductively-coupled plasma mass spectrometry (ICP-MS) have greater sensitivity (˜ppb) than required for meaningful boron distributions for NCT applications (˜tens of ppm). Analytical results are complicated by the potential for cross-contamination between the fractionated cellular components.
Others have attempted to use synchrotron x-ray spectroscopy, secondary ion mass spectrometry (SIMS), and more complex laser-based techniques on dried cells, but all are either very tedious, require demanding and lengthy data analysis, or are of questionable analytical accuracy. None of these techniques can provide the rapid, accurate analyses suitable for statistically meaningful sampling of a large number of cells that is made possible by the present LIBS invention.
Conventional LIBS has been developed for the characterization of biological samples on a macroscopic scale; i.e., a large laser spot size (≧30 μm) with random placement of the beam vaporizing multicellular material. Macroscopic biological applications of LIBS have included trace mineral analyses of skin tissue, fingernails, and teeth, as well as plants and other samples. There have been no reports of LIBS analyses on the subcellular level, and none for the uptake and distribution of pharmaceutical agents. The present invention extends LIBS apparatus capability to single cells on the cellular and subcellular level.